Cell surface receptors transmit signals received on the outside of a cell to the inside through two basic mechanisms: (1) ligand-induced allosteric conformational change and (2) ligand-induced association.
The ligands for the ligand-induced, allosteric conformational change mechanism are typically small molecules, such as the catecholamines or the neuropeptide hormones.
The ligand-induced association mechanism involves an association of specific proteins on the cell surface and has only recently been discovered (relatively speaking), but already has been shown to be as widely used and as important as the first mechanism.
Receptors activated by a ligand-induced dimerization include, for example, those for cell growth and differentiation factors. Factors which serve as ligands for these receptors are typically large polypeptide hormone and cytokines such as erythropoietin, granulocyte colony stimulating factor (G-CSF), or granulocyte macrophage colony stimulating factor (GM-CSF), and human growth hormone (hGH). Many of the dimerization-activated receptors have cytoplasmic tails that contain protein kinase domains or docking sites. Ligand-induced dimerization of the extracellular domains of these receptors results in the juxtaposition of their cytoplasmic tails. They then presumably phosphorylate each other in trans and thereby initiate the cytosolic signaling pathway. In some cases the cytoplasmic domains of dimerization-activated receptors do not have kinase domains themselves, but function the same as if they did because they associate with protein kinases via docking sites.
Receptors activated by oligomerization or aggregation are found most frequently in the immune system. They include, for example, the T cell surface receptors such as CD4, CD8, CD28, CD26, CD45, CD10, and CD3/TCR (T cell antigen receptor). The ligands for these T cell receptors are most often cell surface proteins themselves, and can be found on antigen presenting cells. Aggregation-activated receptors frequently have short cytoplasmic domains which act to bind and thereby recruit other cell surface and/or cytosolic factors following the aggregation of their extracellular domains.
The allosterically activated receptor class has been the primary focus of drug discovery, design, and development efforts for decades. These efforts have yielded many pharmacologically agents. In principle, two distinct types of agents are possible: antagonists and agonists. Antagonists block the binding of the natural ligand without inducing the conformational change in the receptor thereby blocking a signal transduction pathway. Agonists bind to the receptor in a manner which mimics the natural ligand closely enough to induce the same conformational change as natural ligand thereby initiating a signal transduction pathway. See Seed, et al. for a theoretical discussion on how to make an agonist from an antagonist (Seed, B., Making Agonists of Antagonists, Chemistry & Biology 1:125 (1994). See Austin, et al. for a discussion of the role of regulated protein dimerization in biology (Austin, et al. Chemistry & Biology 1:131 (1994)).
Several association-activation receptors have recently become the targets of drug discovery efforts, owing to the important roles they play in various cellular signaling. Low-molecular weight synthetic molecules, that block the interaction of receptors and their ligands and interfere with signal transduction (i.e., antagonists), have been identified using the methods employed with the allosterically activated class. These low-molecular weight synthetic molecules are potential drugs.
Monomeric inhibitors block recall antigen-induced T cell activation and proliferation (G. R. Flentke, E. Munoz, B. T. Huber, A. G. Plaut, C. A. Kettner, and W. W. Bachovchin. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, Proceedings of the National Academy of Sciences of the United States of America 88, 1556-1559 (1991)). A number of anti-CD26 mAbs have the same inhibitory activity when used under non-crosslinking conditions (C. Morimoto, Y. Torimoto, G. Levinson, C. E. Rudd, M. Schrieber, N. H. Dang, N. L. Letvin, and S. F. Schlossman. 1F7, a novel cell surface molecule, involved in helper function of CD4 cells, Journal of Immunology 143, 3430-3439 (1989) and published erratum appears in J. Immunology. 144(5):2027 (March 1990)). Most anti-CD26 mAbs are stimulatory, rather than inhibitory when used under crosslinking conditions (R. W. Barton, J.; Prendergast, and C. A. Kennedy. Binding of the T cell activation monoclonal antibody Ta1 to dipeptidyl peptidase IV, Journal of Leukocyte Biology 48, 291-296 (1990); L. A. Bristol, K, Sakaguchi, E. Appella, D. Doyle, L. Takacs. Thymocyte costimulating antigen is CD26 (dipeptidyl-peptidase IV). Costimulation of granulocyte, macrophage, and T lineage cell proliferation via CD26, Journal of Immunology 149, 367-372. (1992); L. A. Bristol, L. Finch, E. V. Romm, and L. Takacs. Characterization of a novel rat thymocyte costimulating antigen by the monoclonal antibody 1.3, Journal of Immunology 148, 332-338 (1992); B. Fleischer, E. Sturm, V. J. De, and H. Spits. Triggering of cytotoxic T lymphocytes and NK cells via the Tp103 pathway is dependent on the expression of the T cell receptor/CD3 complex, Journal of Immunology 141, 1103-11077 (1988); M. Hegen, G. Niedobitek, C. E. Klein, H. Stein, and B. Fleischer. The T cell triggering molecule Tp103 is associated with dipeptidyl aminopeptidase IV activity, J. Immunol. 144, 2980-2914 (1990)).
A class of low molecular weight synthetic monomeric molecules with high affinity for CD26 have previously been developed and characterized (G. R. Flentke, E. Munoz, B. T. Huber, A. G. Plaut, C. A. Kettner, and W. W. Bachovchin. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, Proceedings of the National Academy of Sciences of the United States of America 88, 1556-1559 (1991); W. G. Gutheil and W. W. Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). These molecules have been shown to be potent and specific synthetic inhibitors for CD26's associated DP IV proteinase activity. DP-IV is a postproline cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of polypeptides.
Representative monomeric structures of these transition-state-analog-based inhibitors, Xaa-boroPro, are e.g., Pro-boroPro and Ala-boroPro. BoroPro refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group [B(OH).sub.2 ]. Pro-boroPro, the most thoroughly characterized of these inhibitors has a Ki of 16 picomolar (pM) (W. G. Gutheil and W. W. Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). Val-boroPro has even a higher affinity, with a Ki of 1.6 pM (W. G. Gutheil and W. W. Bachovchin. Supra; R. J. Snow, W. W. Bachovchin, R. B. Barton, S. J. Campbell, S. J. Coutts, D. M. Freeman, and G. W. Gutheil. Studies on Proline boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic Species Containing a B--N Bond, J. Am. Chem. Soc. 116, 10860-10869 (1994)). Thus, these Xaa-boroPro inhibitors are about 10.sup.+6 fold more potent than the next best known inhibitors. In comparison, antibodies usually have affinities for their targets between 10.sup.-8 and 10.sup.-9 M.
U.S. Pat. Nos. 4,935,493 (the '493 patent) and 5,462,928 (the '928 patent), both of which are incorporated herein by reference, disclose protease inhibitors and transition state analogs (the '493 patent) and methods for treating transplant rejection in a patient, arthritis, or systemic lupus erythematosis (SLE) by administering a potent inhibitor of the catalytic activity of soluble amino peptidase activity of dipeptidyl peptidase type IV (DP-IV; (G. R. Flentke, E. Munoz, B. T. Huber, A. G. Plaut, C. A. Kettner, and W. W. Bachovchin. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, Proceedings of the National Academy of Sciences of the United States of America 88, 1556-1559 (1991)).
Until now, most drug discovery and development efforts have been directed at the allosteric conformational change-activated class of receptors. Also, the efforts directed at the association-activated class have focused on monomeric agents capable of blocking binding of a natural ligand, and therefore blocking signal transduction mediated by these receptors.
Cytotoxic drugs have untoward effects since they indiscriminately kill all proliferating cells. With the advent of monoclonal antibodies, it is possible to increase the specificity of these therapeutic tools. Monoclonal antibodies against the T cell receptors, e.g., T-cell receptor, CD4 and CD8 co-receptors, and to MHC class II molecules, have all been evaluated for their respective benefit in experimental models for the treatment of autoimmune disease. The major impediment to using monoclonal antibodies as a therapeutic tool in humans, is that most monoclonal antibodies are made in mice, and humans rapidly develop an antibody response to mouse antibodies, which limits their potency because of neutralization and, worse, produces allergic reactions such as immune complex disease. Once this has occurred, all mouse monoclonal antibodies become useless in that patient. To avoid this problem, antibodies which are not recognized as foreign by the human immune system are currently being made via three different ways. One approach is to clone human V regions into a phage display library and select for binding to human cells. Using this method, monoclonal antibodies that are entirely human in origin can be obtained. Second, mice that lack endogenous immunoglobulin genes can be made transgenic for human heavy and light chain loci using yeast artificial chromosomes. Third, one may graft the antigen-binding loops of a mouse monoclonal antibody onto the framework of a human immunoglobulin molecule (a process known as humanization).
Each of these three methods produce monoclonal antibodies which are far less immunogenic in humans than the parent mouse monoclonal antibodies, but with each methods comes a host of additional problems or road-blocks. For example, antiidiotype neutralizing antibodies are often produced in patients receiving monoclonal antibody therapy.